Peripheral Blood Polymorphonuclear Cells Research Articles

Metabolic alterations of peripheral blood immune cells and heterogeneity of neutrophil in intracranial aneurysms patients.

Intracranial aneurysms (IAs) represent a severe cerebrovascular disease that can potentially lead to subarachnoid haemorrhage. Previous studies have demonstrated the involvement of peripheral immune cells in the formation and progression of IAs. Nevertheless, the impact of metabolic alterations in peripheral immune cells and changes in neutrophil heterogeneity on the occurrence and progression of IAs remains uncertain. Single-cell Cytometry by Time-of-Flight (CyTOF) technology was employed to profile the single-cell atlas of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) in 72 patients with IAs. In a matched cohort, metabolic shifts in PBMC subsets of IA patients were investigated by contrasting the expression levels of key metabolic enzymes with their respective counterparts in the healthy control group. Simultaneously, compositional differences in peripheral blood PMNs subsets between the two groups were analysed to explore the impact of altered heterogeneity in neutrophils on the initiation and progression of IAs. Furthermore, integrating immune features based on CyTOF analysis and clinical characteristics, we constructed an aneurysm occurrence model and an aneurysm growth model using the random forest method in conjunction with LASSO regression. Different subsets exhibited distinct metabolic characteristics. Overall, PBMCs from patients elevated CD98 expression and increased proliferation. Conversely, CD36 was up-regulated in T cells, B cells and monocytes from the controls but down-regulated in NK and NKT cells. The comparison also revealed differences in the metabolism and function of specific subsets between the two groups. In terms of PMNs, the neutrophil landscape within patients group revealed a pronounced shift towards heightened complexity. Various neutrophil subsets from the IA group generally exhibited lower expression levels of anti-inflammatory functional molecules (IL-4 and IL-10). By integrating clinical and immune features, the constructed aneurysm occurrence model could precisely identify patients with IAs with high prediction accuracy (AUC=0.987). Furthermore, the aneurysm growth model also exhibited superiority over ELAPSS scores in predicting aneurysm growth (lower prediction errors and out-of-bag errors). These findings enhanced our understanding of peripheral immune cell participation in aneurysm formation and growth from the perspectives of immune metabolism and neutrophil heterogeneity. Moreover, the predictive model based on CyTOF features holds the potential to aid in diagnosing and monitoring the progression of human IAs.

Evaluation of peripheral blood polymorphonuclear cell functions after an oral carbohydrate overload in obese and insulin dysregulated horses

Obesity and insulin dysregulation (ID) are increasingly prevalent conditions in equid populations worldwide. Immune impairment is well described in humans with metabolic dysfunction and is reported but still incompletely understood in horses. This study evaluated the effect of acute induced transient hyperglycemia on apoptosis, phagocytosis and oxidative burst activity of peripheral blood polymorphonuclear cells (PMN) of lean and obese adult horses with or without insulin dysregulation. Seventeen adult horses were allocated into three groups based on their body condition score (BCS) and metabolic status: lean-insulin sensitive (lean-IS), obese-insulin sensitive (obese-IS) and obese-insulin dysregulated (obese-ID). ID was determined by insulin tolerance testing (ITT). Blood glucose elevation was induced through an infeed-oral glucose test (in-feed OGT), and all assessments of PMN functions (apoptosis, phagocytosis and oxidative burst) were done in vitro after isolation from peripheral blood before and 120 min after carbohydrate overload. Results were analyzed using a repeated measures linear mixed model with significance defined at P < 0.05. No differences in apoptosis were observed between experimental groups at any time point. Phagocytic capacity was significantly lower at baseline in the obese-ID group but increased in response to glucose administration when compared to the other two groups. Basal reactive oxygen species production in the obese-IS group differed significantly from the lean-IS and obese-ID groups and decreased significantly in response to glucose administration. Results from this study showed that both metabolic status itself, and oral glucose administration, seem to be factors that alter PMN functionality in horses, specifically phagocytosis and oxidative burst.

Human Polymorphonuclear Cells Support Zika Virus to Cross Endothelial Monolayer and Access Bloodstream

The rapid spread of new outbreaks of human infection caused by Zika virus (ZIKV) has raised many global concerns since 2016. Despite the increasing knowledge of this virus, data on the pathogenesis of ZIKV are still missing. In particular, it is still unknown how the virus crosses the endothelial monolayer and gets access to the bloodstream. In the present work, we used human umbilical vein endothelial cells (HUVECs) as a model to study ZIKV infection in vitro. We demonstrated that HUVECs are an optimal reservoir for viral replication, as they were able to sustain ZIKV infection up to two weeks, without showing a cytopathic effect. In order to evaluate the integrity of endothelial monolayer, immunofluorescence was performed on mock-infected or ZIKV-infected cells ± peripheral blood mononuclear cells (PBMCs) or polymorphonuclear cells (PMN), 48 h p.i., by using an anti-VE-Cadherin antibody, a major adherence protein that maintains the integrity of intercellular junctions. In addition to infection, we noted that the presence of some components of the immune system, such as PMNs, played an important role in altering the endothelial monolayer in cell junctions, suggesting that presence at the site of infection probably promotes the spread of ZIKV in vivo in the bloodstream.

Plasma pharmacokinetics, pulmonary disposition, and safety of subcutaneous gamithromycin in alpacas.

The study objective was to determine the disposition of gamithromycin in plasma, peripheral blood polymorphonuclear cells (PMNs), pulmonary epithelial lining fluid (PELF), and bronchoalveolar lavage (BAL) cells in alpacas. A single subcutaneous injection of gamithromycin (6.6mg/kg) was administered to six healthy adult alpacas. At various time points after administration, gamithromycin concentrations were analyzed via LC-MS/MS in plasma, PMNs, PELF, and BAL cells until Day 14 post-injection. Plasma gamithromycin concentrations were measured in all six alpacas; the remaining three body compartments were analyzed in four alpacas. Gamithromycin rapidly concentrated in blood PMNs, BAL cells, and PELF. Shorter Tmax , and lower Cmax, and AUC were observed in plasma than in the other three compartments. Cmax was highest in BAL cells (26001.80±12400.00ng/ml) and PMNs (2573.00±963.30ng/ml) compared to PELF (660.80±413.70ng/ml) and plasma (452.30±196.20ng/ml). Mean terminal half-lives were 72.60±14.10h in plasma, 56.60±10.60h in PELF, 62.80±85.30h in PMNs, and 93.60±124.80h in BAL cells. No injection site reactions occurred. One alpaca developed colic but no other adverse reactions were noted. Overall, gamithromycin was highly concentrated in white blood cells and pulmonary fluids/cells. Clinical utilization of gamithromycin in alpacas should be done with caution until further investigation of potential for colic.

Effect of quercetin on formation of porcine neutrophil extracellular trap

Neutrophil extracellular trap (NET) formation is an immune response to the invasion of external microorganisms. Quercetin, a member of the flavonoid family found in fruits and vegetables, has been examined in multiple biological contexts. The objective of this study was to examine the effect of quercetin on porcine NET formation. We measured NET formation by peripheral blood polymorphonuclear cells (PMNs) using propidium iodide (PI) dye. The amount of tumor necrosis factor (TNF)-α in culture supernatants was quantified by ELISA, and TNF-α mRNA expression was measured by RT-PCR. Direct treatment of PMNs with quercetin did not affect NET formation; however, NET formation was inhibited by exposure to culture supernatant from peripheral blood mononuclear cells (PBMCs) treated with quercetin. By contrast, culture supernatant from PBMCs treated with lipopolysaccharide (LPS) induced high levels of NET formation of PMNs, and this effect was reduced by co-treatment with LPS and quercetin. In addition, treatment of PMNs with recombinant porcine (rp) TNF-α induced high levels of NET formation. PBMCs treated with LPS increased higher levels of TNF-α mRNA and protein, but this effect was weakened when they were co-treated with quercetin. These findings indicated that quercetin inhibits NET formation of PMNs by suppressing production of TNF-α from LPS-stimulated PBMCs. These results suggest that quercetin exerts an anti-inflammatory effect, mediated by down-regulation of TNF-α production from LPS-stimulated PBMCs, which inhibits NET formation in PMNs.

OP0054 NEW ROLE FOR PROTEINASE 3 IN IL-16 BIOACTIVITY CONTROL IN GRANULOMATOSIS WITH POLYANGIITIS

Background:The immunomodulatory cytokine IL-16 is increased in several inflammatory and autoimmune diseases1. IL-16 recruits and activates CD4+ immune cells such as T cells, dendritic cells, or monocytes. IL-16 is produced by various immune and non-immune cells, but synthesis and storage of IL-16 is regulated differentially depending on the cell type and stimulation. For its biological activity, IL-16 cleavage by caspase-3 is required1. Necrotizing granulomatous inflammation is a hallmark of granulomatosis with polyangiitis (GPA) with neutrophil dysregulation as a central driver of chronic inflammation and autoimmunity2. Earlier studies showed a correlation between increased serum IL-16 and disease parameters in AAV, including GPA3, but functional evidence for a direct link between IL-16 and neutrophils in granulomatous inflammation is missing so far.Objectives:In this study we aim to identify a functional link between increased IL-16, neutrophils, and the autoantigen proteinase 3 (PR3) with regard to chronic inflammation and autoimmunity in GPA.Methods:IL-16 was measured in sera of GPA patients (n = 40) and healthy controls (HC, n = 50) by ELISA and correlated with clinical features, such as disease activity (BVAS), creatinine, GFR, VDI and PR3-ANCA status. IL-16 protein expression was analyzed in peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) from GPA patients and HC (n = 5, each) by SDS-PAGE and western blot. Binding affinity of recombinant pro-IL-16 to native human PR3 was assessed by microscale thermophoresis. Cleavage of pro-IL-16 by active human PR3 was performed at various time points at 37°C. Cleavage products were analyzed by SDS-PAGE and western blot.Results:Circulating IL-16 was significantly increased in GPA patients compared to HC. Elevated IL-16 positively correlated with BVAS, creatinine, VDI and PR3-ANCA status and negatively correlated with GFR. In PMBC and PMN from GPA and HC we identified different expression patters of precursor and active forms of IL-16. In healthy PBMC we found high amounts of precursor (80kD), pro-IL-16 (55kD) and active IL-16 (17kD). In contrast, PBMC from GPA patients had lower amounts of pro-IL-16 and no active IL-16, indicating activation and secretion of IL-16 due to inflammatory stimulation, as shown earlier5. In GPA PMN we detected no precursor IL-16, but pro-IL-16 and its active form, in contrast to very low amounts of all IL-16 forms in healthy PMN. Processing and release of IL-16 in neutrophils has been linked to apoptosis and secondary necrosis5. By interaction studies we demonstrated direct binding of pro-IL-16 to PR3 with a Kd of 10 nM. In a subsequent cleavage assay we confirmed IL-16 processing by PR3 in a time-dependent manner.Conclusion:Correlation of serum IL-16 with clinical features of GPA suggests that IL-16 is associated with markers of disease activity, tissue damage and autoreactivity. We showed that PBMC and PMN represent a source of IL-16 in GPA. By the identification of PR3 as an additional IL-16-activating enzyme we could demonstrate a potential link between excessive PR3 expression, cell death and IL-16-dependent mechanisms, contributing to chronic granulomatous inflammation and autoimmunity in GPA.

Regulation of toll-like receptors expression in muscle cells by exercise-induced stress.

ObjectiveThis study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them.MethodsThe expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H2O2), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells.ResultsThe qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H2O2, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells.ConclusionIn this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H2O2, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

Mutated GM-CSF-Based CAR T-Cells Targeting CD116/CD131 Complexes Exhibit Enhanced Anti-Tumor Effects Against Acute Myeloid Leukemia

Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%-80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads. Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p &amp;lt; 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p &amp;lt; 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for &amp;gt; 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal. Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells. Disclosures Saito: Toshiba Corporation: Research Funding. Hasegawa:Toshiba Corporation: Research Funding. Inada:Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima:Toshiba Corporation: Research Funding. Yagyu:Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

Zinc Enhances Neutrophil Extracellular Trap Formation of Porcine Peripheral Blood Polymorphonuclear Cells through Tumor Necrosis Factor-Alpha from Peripheral Blood Mononuclear Cells

Zinc Enhances Neutrophil Extracellular Trap Formation of Porcine Peripheral Blood Polymorphonuclear Cells through Tumor Necrosis Factor-Alpha from Peripheral Blood Mononuclear Cells

Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

CFTR Modulators Dampen Aspergillus-Induced Reactive Oxygen Species Production by Cystic Fibrosis Phagocytes.

Excessive inflammation by phagocytes during Aspergillus fumigatus infection is thought to promote lung function decline in CF patients. CFTR modulators have been shown to reduce A. fumigatus colonization in vivo, however, their antifungal and anti-inflammatory mechanisms are unclear. Other treatments including azithromycin and acebilustat may dampen Aspergillus-induced inflammation due to their immunomodulatory properties. Therefore, we set out in this study to determine the effects of current CF therapies on ROS production and fungal killing, either direct or indirect by enhancing antifungal immune mechanisms in peripheral blood immune cells from CF patients upon A. fumigatus infection. Isolated peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) from CF patients and healthy volunteers were challenged with A. fumigatus following pre-treatment with CFTR modulators, azithromycin or acebilustat. Ivacaftor/lumacaftor treated CF and control subject PMNs resulted in a significant reduction (p < 0.05) in Aspergillus-induced ROS. For CF PBMC, Aspergillus-induced ROS was significantly reduced when pre-treated with ivacaftor alone (p < 0.01) or in combination with lumacaftor (p < 0.01), with a comparable significant reduction in control subject PBMC (p < 0.05). Azithromycin and acebilustat had no effect on ROS production by CF or control subject phagocytes. None of the treatments showed an indirect or direct antifungal activity. In summary, CFTR modulators have potential for additional immunomodulatory benefits to prevent or treat Aspergillus-induced inflammation in CF. The comparable effects of CFTR modulators observed in phagocytes from control subjects questions their exact mechanism of action.

Nickel Increases Chemotactic Activity of Porcine Peripheral Blood Polymorphonuclear Cells

Nickel is a nutritionally essential trace element that plays an important role in the immune system of several animal species. The aim of this study was to examine the effect of nickel chloride on chemotactic activity of peripheral blood polymorphonuclear cells (PMNs) and whether this effect is associated with interleukin (IL)-8 and a nuclear factorkappa B (NF-κB)-dependent pathway. Peripheral blood mononuclear cells (PBMCs) and PMNs were isolated by Percoll solution (Specific gravity; 1.080) and 1.5% dextran treatment, respectively. A modified Boyden chamber assay was used to measure the chemotactic activity of PMNs. The level of IL-8 in culture supernatant from PBMCs was measured by enzyme-linked immunosorbent assay (ELISA). Both of PBMCs and PMNs exhibited a low viability when cultured with concentration of greater than 1,000 µM of nickel chloride for 24 h. Thus, nickel chloride was used at concentration of 500 µM, which preserved cell viability. Treatment with nickel did not directly affect the chemotactic activity of PMNs. However, the chemotactic activity of PMNs was remarkably increased by culture supernatant from PBMCs treated with nickel chloride (500 µM) for 24 h. Recombinant porcine IL-8 polyclonal antibody (pAb) neutralized the enhancing effect on the chemotactic activity of PMNs by culture supernatant from PBMCs treated with nickel and this culture supernatant had higher IL-8 levels than the culture supernatant from untreated PBMCs. In addition, n-tosyll-phenylalanine chloromethyl ketone (TPCK), a NF-κB inhibitor, antagonized the enhancing effect on the chemotactic activity of PMNs by the culture supernatant from PBMCs treated with nickel. These results suggested that nickel stimulates porcine PBMCs to produce IL-8, which increases the chemotaxis of PMNs via NF-κB-dependent pathway.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

PSXIII-18 Conceptus stimuli in peripheral blood mono and polymorphonuclear cells at the beginning of pregnancy

Abstract The objective of this experiment was to compare the expression of Interferon-tau Stimulated Genes (ISGs) in peripheral blood mono and polymorphonuclear cells (PBMCs and PMNs) in heifers following insemination. Twenty-nine Nelore heifers had estrous cycle synchronized, and FTAI occurred on D0. Pregnancy diagnosis was performed by ultrasonography on D28 post FTAI. On D0, 10, 14, 16, 18 and 20, blood (25mL) from the jugular vein was collected in heparinized tubes for isolation of PMNs and PBMCs. The isolation was performed using Ficoll®Paque Plus (GE Healthcare). PMNs and PBMCs samples from 8 pregnant and 9 non-pregnant heifers were subjected to RNA extraction using the DirectZol-RNA kit (Zymo-Research) and Trizol (Invitrogen), respectively. The expression of the target genes (ISG15, OAS-1, MX1 and MX2) was normalized in relation to the two reference genes (GAPDH/ACTB for PMNs and GAPDH/PPIA for PBMCs). The abundance of transcripts was evaluated by analysis of variance considering fixed effects of group, day and group by day interaction using the PROC MIXED procedure in SAS. PMNs and PBMCs had a similar expression profile of ISG15 and OAS-1, showing a relative increase (P &amp;lt; 0.05) from D18, and a significant increase in (P &amp;lt; 0.05) expression in pregnant compared to non-pregnant females on D18 and D20. These results were also observed for MX1 in PBMCs. In PMNs, no significant effects for MX1 were found. For MX2, in both cells types, only a group effect (P &amp;lt; 0.05) was observed, indicating a higher expression in pregnant heifers (0.57±0.11 vs. 0.21±0.03) on the days evaluated. When comparing the relative expression of the target genes to D0, no significant (P &amp;gt; 0.1) differences were found between PMNs and PBMCs. In summary, ISG expression is similar in PMNs and PBMCs, specifically for ISG15 and OAS-1, which both seem to be suitable biomarkers for potential early pregnancy determination in heifers.

64 Prediction of pregnancy and early embryo loss through OAS-1 expression, concentrations of pregnancy-associated glycoproteins, and Doppler ultrasonography in beef cattle

We aimed to compare three methods to detect pregnancy based on interferon-tau stimulated gene expression in peripheral blood polymorphonuclear cells and Doppler ultrasonography (Doppler-US) 20 days after timed AI and the concentrations of pregnancy associated glycoproteins (PAGs) 25 days after timed AI. Our second objective was to compare interferon-tau stimulated genes and PAGs as early embryo loss (EEL) predictors. Nelore cows (n=144) and heifers (n=103) were submitted to timed AI (Day (D) 0). On D20, polymorphonuclear cells were isolated from blood samples by Ficoll gradient (GE Healthcare), and the RNA was extracted. Expression of the interferon-tau stimulated gene (OAS-1) was quantified by qPCR and normalized to reference genes (GAPDH and ACTB). On D25, blood was collected from the jugular vein and PAG concentrations were measured by an in-house enzyme-linked immunosorbent assay. Pregnancy diagnoses were performed on D20 by luteolysis detection with Doppler-US and on D30 by detection of an embryo by heartbeat. Animals were classified as pregnant (P; fetus on D30), non-P (NP; no active corpus luteum on D20), and EEL (active corpus luteum on D20 but NP on D30). Expression of OAS-1 and PAG concentrations were analysed by analysis of variance using PROC MIXED of SAS considering the effects of group, category, and their interaction. Receiver operating characteristic curves were created, and the area under the curve (AUC), accuracy, specificity, and sensitivity were calculated for pregnancy predictions on D20 (OAS-1 and Doppler-US) and D25 (PAGs) compared with the standard diagnosis method on D30. Expression of OAS-1 and PAG concentrations were greater (P&amp;lt;0.01) in the P (2.4±0.2 and 4.8±0.2ngmL−1, respectively) compared with the NP (0.50±0.1 and 1.2±0.2ngmL−1, respectively) and EEL (0.8±0.2 and 1.0±0.2ngmL−1, respectively) groups. Receiver operating characteristic analysis indicated that OAS-1, Doppler, and PAGs were significant (P&amp;lt;0.01) predictors of pregnancy in heifers (AUC=0.86, 0.92, and 0.94, respectively) and cows (AUC=0.82, 0.94, and 0.95, respectively). The PAGs and Doppler-US presented higher accuracy on diagnosing pregnancy (92 and 89% for heifers, 94 and 93% for cows, respectively) than OAS-1 (81% for heifers and 75% for cows). Doppler-US was 100% sensitive to detect nonpregnant females, which avoided false negative results, whereas PAG concentrations presented a higher specificity (86% for heifers and cows) when compared with Doppler-US and OAS-1 (76 and 65% for heifers, 85 and 75% for cows, respectively). On D25, PAGs were 90% accurate to detect heifers and cows that would experience EEL, whereas on D20, OAS-1 was 50% accurate. In conclusion, Doppler-US can be used as early as D20 with high accuracy and sensitivity. Likely, the use of PAG concentrations on D25 is highly accurate and is more effective at detecting EEL. Pregnancy diagnosis through OAS-1 expression is not as accurate as the other methods and could not efficiently predict EEL. This research was supported by FAPESP (2015/106069; 2017/134729).

Dynamics of white and red blood cells in the ontogenesis of African catfish

The paper presents the results of the study of age dynamics and sexual characteristics of blood cells. The object of the study was African catfish during puberty (six months, one year and two years old). At the first stage, the age dynamics of the red blood cells content was studied. The number of red blood cells in the blood of African catfish, depending on age, varied in the range from 0.85*1012 cells/l to 1.35*1012 cells/l. As puberty, the content of red blood cells increased. Erythropoiesis peaked and stabilized at the age of two years. The content of white blood cells of peripheral blood with age increasing twice, reaching a maximum in adulthood-1.14*109 cells/l in females and 1.01*109 cells/l in males. Analysis of the leukocyte formula of African catfish revealed age-dependent quantitative redistribution of cells. It was found in the course of studies, that with age, the proportion of white blood cells in the leukocyte formula of the African catfish increases. There is a general tendency to reduce the relative content of neutrophils. The dynamics of indicators is most clearly manifested in the critical periods of puberty of fish. Studies have shown that a pronounced predominance in the leukocyte formula of white blood cells (within the physiological norm), persists in all age periods and is characteristic of females and males. Age redistribution of leukocyte cells series: neutrophils, monocytes and polymorphonuclear cells in peripheral blood of African catfish demonstrates the development and formation of cellular and humoral links of the immune system in ontogenesis. In the implementation of this process, males and females clearly manifest sexual differences, which become more pronounced with the age of fish.

Comparison for immunophysiological responses of Jeju and Thoroughbred horses after exercise.

ObjectiveThe study was conducted to investigate variations in the immunophysiological responses to exercise-induced stress in Jeju and Thoroughbred horses.MethodsBlood samples were collected from the jugular veins of adult Jeju (n = 5) and Thoroughbred (n = 5) horses before and after 30 min of exercise. The hematological, biochemical, and immunological profiles of the blood samples were analyzed. Blood smears were stained and observed under a microscope. The concentration of cell-free (cf) DNA in the plasma was determined using real time polymerase chain reaction (PCR). Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells were separated using Polymorphprep, and the expression of various stress-related and chemokine receptor genes was measured using reverse transcriptase (RT) and real-time PCR.ResultsAfter exercise, Jeju and Thoroughbred horses displayed stress responses with significantly increased rectal temperatures, cortisol levels, and muscle catabolism-associated metabolites. Red blood cell indices were significantly higher in Thoroughbred horses than in Jeju horses after exercise. In addition, exercise-induced stress triggered the formation of neutrophil extracellular traps (NETs) and reduced platelet counts in Jeju horses but not in Thoroughbred horses. Heat shock protein 72 and heat shock protein family A (Hsp70) member 6 expression is rapidly modulated in response to exercise-induced stress in the PBMCs of Jeju horses. The expression of CXC chemokine receptor 4 in PBMCs was higher in Thoroughbred horses than in Jeju horses after exercise.ConclusionIn summary, the different immunophysiological responses of Jeju and Thoroughbred horses explain the differences in the physiological and anatomical properties of the two breeds. The physiology of Thoroughbred horses makes them suitable for racing as they are less sensitive to exercise-induced stress compared to that of Jeju horses. This study provides a basis for investigating the link between exercise-induced stresses and the physiological alteration of horses. Hence, our findings show that some of assessed parameters could be used to determine the endurance performance of horses.

Proteomic analysis of peripheral blood polymorphonuclear cells (PBMCs) reveals alteration of neutrophil extracellular trap (NET) components in uncontrolled diabetes

Neutrophils have been thought to play a major role in inflammation and diabetic complications especially in poor glycemic control patients as demonstrated by their aberrant inflammatory markers. The aim of the present study was to compare neutrophil proteome profiles between diabetic patients with good glycemic control and those with poor glycemic control to see whether there might be any differences that could be related to the cause of complications which are found more commonly in the latter. Using 2-dimensional gel electrophoresis (2-DE) followed by quadrupole time of flight mass spectrometry (Q-TOF MS) and/or tandem mass spectrometry (MS/MS), we identified 35 differentially expressed proteins, some of which were protein components of neutrophil extracellular traps (NETs), in the poor glycemic control group compared to the good glycemic control group. The observed alterations of protein components of NETs included downregulation of myeloperoxidase, azurocidin (CAP37), and S100A9; and upregulation of the glycolytic enzymes transketolase and alpha-enolase. Manganese superoxide dismutase (MnSOD), functioning in cellular response and defense, was also found downregulated in the poor control group. Most of the glycolysis-related proteins were downregulated in the good control group but upregulated in the poor control group, including phosphoglycerate kinase 1 (PGK1) and L-lactate dehydrogenase B chain (LDHB). The findings of this study demonstrate the dysregulation of protein components of NETs in neutrophils in patients with poorly controlled diabetes. More specifically, these findings suggest association between NETs and inflammation in diabetes and provide further insights into the role of neutrophils in the complications of poorly controlled diabetes.

Effects of Light on In Vitro Production of Melatonin by Human Peripheral Blood Mononuclear, Polymorphonuclear, and Whole Blood Cells

Recent studies have shown that melatonin can be secreted, in addition to other sources, by peripheral blood mononuclear cells (PBMCs). We investigated whether melatonin could also be secreted by cultured polymorphonuclear (PMNs) and whole blood (WB) cells under resting and stimulated conditions. In addition, influences of the intensity and color of light on the production of melatonin by these cells were estimated. Blood samples were collected from a healthy volunteer, and PBMCs and PMNs were isolated. The PBMC, PMN, and WB cultures were exposed for 8 h to light intensities of 0, 20, 50, 100, 300, and 500 lux and to white, red, blue, and green lights in the presence/absence of mitogens. Levels of melatonin in supernatants were detected by enzyme-linked immunosorbent assay (ELISA). In addition to PBMCs, PMNs and WB cells were found to also produce melatonin, and this production was increased due to mitogen activations. The most effective intensity of light, which suppresses melatonin secretion, was 100 lux. There were no significant differences between the influences of different monochromatic lights on the melatonin production by the examined blood cell cultures. Thus, PBMCs, PMNs, and WB cultures can produce melatonin, and mitogen activation causes a higher production of this hormone. Moreover, although white light suppressed melatonin secretion at 100 lux, there was no difference among various types of lights. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

α-2,3-Sialyltransferase 1 and neuraminidase-3 from monocytes in patients with rheumatoid arthritis correlate with disease activity measures: A pilot study.

We decided to study the association of monocyte α-2,3-sialyltransferase 1 (ST3Gal-1), neuraminidase-3 (Neu3), α-2,6-sialyltransferase 1 (ST6Gal-1), and neuraminidase-1 (Neu1) levels with disease activity score 28 (DAS28) in human rheumatoid arthritis (RA), considering that mouse monocytes' sialic acid (SIA) levels relate to their phagocytosis and IgG binding ability. ST3Gal-1, Neu3, ST6Gal-1, Neu1, α-2,3-SIA, and α-2,6-SIA levels on RA peripheral blood monocytes, T cells, and polymorphonuclear cells were determined by using fluorochrome-conjugated anti-cell-specific marker antibodies and fluorochrome-conjugated anti-enzyme antibodies. Simple correlation and linear regression were used to correlate enzyme levels with DAS28. RA monocyte ST3Gal-1 and Neu3 levels correlated with DAS28 in patients having DAS28 >5.1 (r = 0.469, p = 0.002; r = 0.410, p = 0.006, respectively). When multivariable analysis was performed for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and SIA-related enzyme levels in different cell types as independent variables with DAS28 as a dependent variable, monocyte ST3Gal-1 levels correlated with DAS28 (p = 0.009) but not ESR and CRP in patients having DAS28 >5.1 (both p ≥ 0.292). RA monocyte ST3Gal-1 levels correlated with DAS28 (p = 0.010) and with ESR (p < 0.001) at month 0 when applied to all RA patients including both remission and nonremission groups in multivariable analysis. The latter findings persisted longitudinally at month 3. Monocyte ST3Gal-1 and Neu3 levels correlated longitudinally with DAS28 by two different methods suggest that monocyte ST3Gal-1 and Neu3 levels may be used as biomarkers to monitor RA disease activity.